USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells

Aidan P McCann, Peter Smyth, Francesco Cogo, William J McDaid, Lai Jiang, Jia Lin, Emma Evergren, Roberta E Burden, Sandra Van Schaeybroeck, Christopher J Scott, James F Burrows

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Abstract

BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs).

METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed.

RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib.

CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

Original languageEnglish
Pages (from-to)77
JournalCell communication and signaling : CCS
Volume16
Issue number1
DOIs
Publication statusPublished - 08 Nov 2018

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Clathrin
Protein-Tyrosine Kinases
Exons
Chemical activation
Apoptosis
Trypan Blue
Confocal microscopy
Mutation
Endocytosis
Tumors
Assays
Phosphotransferases
Cells
Growth
Confocal Microscopy
Cell Cycle
Western Blotting
gefitinib
Neoplasms

Cite this

@article{83f79ef310db44e8821cec44d31e34bd,
title = "USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells",
abstract = "BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs).METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed.RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib.CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.",
author = "McCann, {Aidan P} and Peter Smyth and Francesco Cogo and McDaid, {William J} and Lai Jiang and Jia Lin and Emma Evergren and Burden, {Roberta E} and {Van Schaeybroeck}, Sandra and Scott, {Christopher J} and Burrows, {James F}",
year = "2018",
month = "11",
day = "8",
doi = "10.1186/s12964-018-0291-5",
language = "English",
volume = "16",
pages = "77",
journal = "Cell communication and signaling : CCS",
issn = "1478-811X",
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TY - JOUR

T1 - USP17 is required for trafficking and oncogenic signaling of mutant EGFR in NSCLC cells

AU - McCann, Aidan P

AU - Smyth, Peter

AU - Cogo, Francesco

AU - McDaid, William J

AU - Jiang, Lai

AU - Lin, Jia

AU - Evergren, Emma

AU - Burden, Roberta E

AU - Van Schaeybroeck, Sandra

AU - Scott, Christopher J

AU - Burrows, James F

PY - 2018/11/8

Y1 - 2018/11/8

N2 - BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs).METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed.RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib.CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

AB - BACKGROUND: The deubiquitinase USP17 is overexpressed in NSCLC and has been shown to be required for the growth and motility of EGFR wild-type (WT) NSCLC cells. USP17 is also required for clathrin-mediated endocytosis of EGFR. Here, we examine the impact of USP17 depletion on the growth, as well as EGFR endocytosis and signaling, of EGFR mutant (MT) NSCLC cells. In particular, we examine NSCLC cells harboring an EGFR activating exon 19 deletion (HCC827), or both the L858R activating mutation and the T790M resistance gatekeeper mutation (H1975) which renders them resistant to EGFR tyrosine kinase inhibitors (TKIs).METHODS: MTT, trypan blue and clonogenic assays, confocal microscopy, Western blotting and cell cycle analysis were performed.RESULTS: USP17 depletion blocks the growth of EGFRMT NSCLC cells carrying either the EGFR exon 19 deletion, or L858R/T790M double mutation. In contrast to EGFRWT cells, USP17 depletion also triggers apoptosis of EGFRMT NSCLC cells. USP17 is required for clathrin-mediated endocytosis in these EGFRMT NSCLC cells, but it is not required for the internalization of the mutated EGFR receptors. Instead, USP17 depletion alters the localization of these receptors within the cell, and although it does not decrease basal EGFR activation, it potently reduces activation of Src, a key kinase in mutant EGFR-dependent tumorigenicity. Finally, we demonstrate that USP17 depletion can trigger apoptosis in EGFRWT NSCLC cells, when combined with the EGFR tyrosine kinase inhibitor (TKI) gefitinib.CONCLUSIONS: Our data reveals that USP17 facilitates trafficking and oncogenic signaling of mutant EGFR and indicates targeting USP17 could represent a viable therapeutic strategy in NSCLC tumours carrying either an EGFR activating mutation, or a resistance gatekeeper mutation.

U2 - 10.1186/s12964-018-0291-5

DO - 10.1186/s12964-018-0291-5

M3 - Article

C2 - 30409180

VL - 16

SP - 77

JO - Cell communication and signaling : CCS

JF - Cell communication and signaling : CCS

SN - 1478-811X

IS - 1

ER -